Nucleotide sequence of threonine operon irrepressible by isoleucine and method for producing L-threonine using transformed host cell containing the same

ABSTRACT

A nucleotide sequence of the threonine operon of  E. coli  with a deletion of all or part of a nucleotide fragment of −56 to −18, a recombinant vector containing the above nucleotide sequence, and a transformed host cell containing the recombinant vector are provided. A method for producing L-threonine comprising culturing the transformed host cell is also provided.

TECHNICAL FIELD

The present invention relates to a nucleotide sequence of the threonineoperon of Escherichia coli with a deletion of all or part of anucleotide fragment of −56 to −18, a recombinant vector containing theabove nucleotide sequence, and a transformed host cell containing therecombinant vector. The present invention also provides a method forproducing L-threonine comprising culturing the transformed host cell.

BACKGROUND ART

L-threonine is an essential amino acid and is widely used as a feed orfood additive. In addition, L-threonine is used as a medical solution ora raw material for a drug synthesis.

L-threonine is produced by a fermentation process using a mutant strainderived from a wild-type strain of Escherichia coli (E. coli),Corynebacteria sp., Serratia sp., or Providencia sp. Examples of themutant strain include an amino acid analogue- or drug-resistant mutantstrain, or a variety of auxotroph to diaminopimeric acid, methionine,lysine, or isoleucine [Japanese Patent Laid-Open Publication No. Hei.2-219582; Appl. Microbiol. Biotechnol., 29. 550-553 (1988); KoreanPatent No. 1992-8365].

A fermentation process using a recombinant strain can also be used toproduce L-threonine. Japanese Patent Laid-Open Publication No. Hei.5-10076 discloses a method for producing threonine in large scale usinga recombinant strain of Serratia sp. containing a DNA fragment withgenetic information of aspartokinase, homoserine kinase, homoserinedehydrogenase, and threonine synthase. In addition, a method formass-producing L-threonine using a gene derived from a strain ofProvidencia sp. resistant to antagonist of methionine metabolism isdisclosed in Japanese Patent Laid-Open Publication No. Hei. 1-289493.

As a method for producing threonine by regulation of expression ofthreonine operon, replacing a promoter of the threonine operon with tacpromoter (WO 98/04715) and replacing an expression regulatory region ofthe threonine operon with cl repressor and PR promoter of E. coli λphage (EP 0593792B1) are disclosed.

The threonine biosynthesis operon of E. coli is composed of genes, thrA,thrB, and thrC. The thrA gene encodes for aspartokinase and homoserinedehydrogenase, the thrB gene encodes for homoserine kinase, and the thrCgene encodes for threonine synthase. A leader sequence and attenuator ofthese genes precedes the threonine operon [Proc. Natl. Acad. Sci.America (1979), 76: 1706-1710]. One example of the nucleotide sequenceof the threonine operon comprising the leader sequence, the attenuator,and the structural thr genes is as set forth in SEQ ID NO: 1. The ATGcodon of nucleotide positions 337-339 of SEQ ID NO: 1 is a start codonfor the threonine operon. In particular, the expression of the threonineoperon is regulated by the intracellular levels of both threonine andisoleucine. This is similar to the regulatory mechanism of thetryptophan operon [J. Bacteriology (1975), 161-166].

DISCLOSURE OF THE INVENTION

For the threonine-producing strain that is not repressed by threonine orisoleucine, the present inventors have achieved the goals of the presentinvention by providing the mutant threonine operon which contains adefective attenuator region.

Therefore, the present invention is to provide a nucleotide sequence ofthe threonine operon that is not repressible by isoleucine.

The present invention is also to provide a recombinant vector containingthe above nucleotide sequence of the threonine operon.

The present invention is also to provide a transformed host cellcontaining the recombinant vector.

The present invention is further to provide a method for producingL-threonine comprising culturing the transformed host cell.

According to an aspect of the present invention, there is provided anucleotide sequence of the threonine operon of E. coli with a deletionof all or part of a 39 bp nucleotide fragment of −56 to −18 that isattenuator of the structural genes of the threonine operon. In thiscase, the nucleotide sequences in an upstream region from the A of thestart codon, ATG (+1) are numbered as negative numbers, and thenucleotide sequences in a downstream region as positive numbers. Unlessspecified otherwise, the nucleotide numbering is as defined in theabove.

According to another aspect of the present invention, there is provideda recombinant vector containing the above nucleotide sequence of thethreonine operon of E. coli. The recombinant vector can be manufacturedby a conventional method comprising digesting the threonine operon of E.coli and a suitable vector with restriction enzymes such as ApaI andPstI, followed by ligation. The suitable vector may be phage, plasmid,and cosmid, for examples. Examples of the phage and cosmid vectorsinclude pWE15, M13, λEMBL3, λEMBL4, λFIXII, λDASHII, λZAPII, λgt10,λgt11, Charon4A, and Charon21A.

Examples of the plasmid vector include pBR series, pUC series,pBluescriptII series, pGEM series, pTZ series, and pET series. Inaddition, various shuttle vectors that can replicate in a variety ofhost cells such as E. coli and Corynebacteria may be used. Preferably,pECCG122 (KFCC 10696), which has been deposited the Korean Federation ofCulture Collection (KFCC) on Jun. 20, 1990, is used as the cloningvector.

According to another aspect of the present invention, there is provideda novel microorganism containing the above recombinant vector. Themicroorganism may be manufactured by transforming a host cell with therecombinant vector using a conventional method [Sambrook, J. et al., 2th(1989), Cold Spring Harbor Laboratory Press].

A host cell may be gram-negative bacteria. Cells belong to the genusEscherichia sp. are most preferred. In particular, according to thepreferred embodiment of the present invention, E. coli KCCM 10236(Korean Patent Application No. 2001-6976) is used as a host cell. The E.coli KCCM 10236 is an auxotroph for methionine and is resistant tothreonine analogue (AHV: α-amino-β-hydroxyvaleric acid), lysine analogue(AEC: S-(2-aminoethyl)-L-cysteine), isoleucine analogue (α-aminobutyricacid), and methionine analogue (ethionine). In addition, the E. coliKCCM 10236 has an extra copy of phosphoenol pyruvate carboxylase gene ina chromosomal DNA and at least one of the thrA, thrB, and thrC genes ispresent in an amplified state, thereby its productivity of L-threonineis increased. In the present invention, the effects of the aboverecombinant vector were confirmed by using Escherichia coli KFCC 10236as a host cell. The Escherichia coli KFCC 10236 has been deposited inthe Korean Federation of Culture Collection (KFCC) on Dec. 29, 2000.

According to yet another aspect of the present invention, there isprovided a method for producing L-threonine comprising culturing thetransformed host cell and recovering L-threonine from the culture.Preferably, E. coli KCCM 10236/pTHR(+) is used as the transformed hostcell.

The method for producing L-threonine of the present invention may becarried out under a conventional condition. Although the intracellularlevels of isoleucine and threonine are high, the production ofL-threonine is not inhibited. Thereby the productivity of L-threonine isincreased.

Hereinafter, the present invention will be described more specificallyby examples. However, the following examples are provided only forillustrations and thus the present invention is not limited to or bythem.

EXAMPLES

Example 1 showed correlation between isoleucine concentration in aculture medium and L-threonine biosynthesis using a conventionalL-threonine-producing strain. The nucleotide sequence of the threonineoperon (hereinafter, simply referred to as “the thr operon”) was clonedfrom the strain of Example 1 (Example 2). Then, mutation was induced onthe pTHR (−) vector containing the cloned thr operon (Example 3) andstrains of which the expression of the thr operon cannot be repressed byisoleucine and threonine were selected (Example 4). Finally, in Example5, the thr operon of the selected strains was sequenced. As a result, apart of the attenuator in the thr operon was deleted.

Example 1 Correlation Between Isoleucine Concentration in Culture Mediumand L-threonine Biosynthesis

E. coli KCCM 10236 strains were fed-batch cultured in a fermenter withcapacity of 30 liter for 77 hours while maintaining a culturetemperature of 32° C., a culture pH of 6.5, and aeration rate of 0.5 to1.0 vvm. The composition and concentration of amino acids in the finalculture medium were determined by an automatic amino acid analyzer. Theresults are presented in Table 1.

TABLE 1 Composition and concentration of amino acids in E. coli KCCM10236 culture medium Amino acid Concentration (g/l) Amino acidConcentration (g/l) Aspartate 0.00 Alanine 0.10 Glutamate 1.20 Tyrosine0.00 Asparagine 0.00 Methionine 0.38 Serine 0.07 Valine 0.73 Glutamine0.00 Phenylalanine 0.33 Histidine 0.00 Isoleucine 0.67 Glycine 0.00Leucine 0.59 Threonine 115.20 Lycine 0.00 Arginine 0.00

In order to investigate the productivity of L-threonine depending on theadded amount of L-isoleucine, 0 to 0.4 g/l of L-isoleucine was added tofermentation media and the resultant media were incubated in a flask for48 hours. The results are shown in Table 2.

TABLE 2 Effect of addition of L-isoleucine on productivity ofL-threonine of E. coli KCCM 10236 amount of concentration ofProductivity of L-threonine L-isoleucine (g/l) L-threonine (g/l) (%) 018.7 37.4 0.05 16.8 33.6 0.10 9.52 19.0 0.20 8.26 16.5 0.40 8.35 16.7

As shown in Table 2, in the case of a conventional threonine productionstrain, E. coli KCCM 10236, as increasing the amount of L-isoleucine,the productivity of L-threonine was rapidly reduced.

Example 2 Cloning of Nucleotide Sequence of thr Operon in E. coli KCCM10236

The thr operon was amplified and cloned from E. coli KCCM 10236. Primersfor amplification of the thr operon were thr-F and thr-R as set forth inSEQ ID NO: 3 and SEQ ID NO: 4, respectively. The primer sequences weredesigned using the GenBank database (NCBI).

5447 bp of the thr operon containing promoter region was amplified usingExpand™ High Fidelity PCR System (Boehringer Mannheim). The PCR reactionmixture contained 200 μM of each dNTP, 300 nM of each primer, 0.1 μg ofE. coli KCCM 10236 genomic DNA, 1× Expand HF buffer with 15 mM MgCl₂,and 2.6 U of Expand™ High Fidelity PCR System enzyme mix. The PCRreaction was performed at 95° C. for 5 minutes, 95° C. for 30 seconds,56° C. for 30 seconds, and 68° C. for 4 minutes and 30 seconds for 30cycles to amplify the thr operon. The PCR reaction was maintained at 68°C. for 10 minutes and then terminated at 4° C. The amplified thr operonwas inserted into a cloning vector, pGEM-T (Promega, America). Then, E.coli DH5a was transformed with the cloning vector containing the throperon to obtain a pGEM-Thr vector.

The pGEM-Thr and pECCG 122 (KFCC 10696) vectors were digested withrestriction enzymes, ApaI and PstI, and ligated with the thr operon andpECCG122 vector using a conventional method. E. coli DH5a wastransformed with the ligation product to obtain a pTHR(−) vector.According to sequencing analysis, the cloned thr operon contained thenucleotide sequence of a conventional thr operon as set forth in SEQ IDNO: 1.

Example 3 Mutation of THR(−)

In order to induce mutation on the thr operon, 2 μg of the pTHR(−) DNAcontaining the thr operon was dissolved in 200 μl of buffer (0.1MKH₂PO₄-1 mM EDTA, pH 6.0) containing 200-500 μg/ml ofN-methyl-N′-nitro-N-nitrosoguanidine and incubated at 37° C. for 10-30minutes. The mutation-induced vector was recovered using a conventionalalcohol precipitation and E. coli W3110 (ATCC27325) was transformed withthe recovered vector. Then, the transformed E. coli W3110 culture wasplated on a LB medium containing 50 mg/l of kanamycine to obtaincolonies grown on the LB medium. The colonies were replica plated onto aselective minimal medium plate containing isoleucine hydroxamate as anisoleucine analogue and α-amino-β-hydroxyvaleric acid (AHV) as athreonine analogue and onto a control minimal medium plate withoutcontaining these analogues, Strains grown onto the selective minimalmedium plate were selected. After the replica plating was repeated threetimes, 27 candidate strains were selected. Then, the selected candidatestrains were again inoculated onto selective minimal media platecontaining isoleucine hydroxamate as an isoleucine analogue andα-amino-β-hydroxyvaleric acid (AHV) as a threonine analogue to therebyreconfirm the growth of the candidate strains on the selective minimalmedia plate.

Example 4 Determination of Nucleotide Sequence of thr OperonIrrepressible by Isoleucine and Resistance of thr Operon to Isoleucine

Mutation-induced pTHR(*) vectors were recovered using a conventionalmethod from the 27 candidate strains obtained in Example 3 and then athreonine-producing strain, E. coli KCCM 10236 was transformed with thepTHR(*) vectors. The recombinant E. coli KCCM 10236 strains were platedonto LB media containing 50 mg/l of kanamycin to obtain colonies grownon the LB media. The obtained recombinant strains containing the pTHR(*)vectors, total 135 strains were cultured on solid media of Table 3.Then, one loopful of each culture was inoculated in a flask containingthe fermentation medium of Table 4 and cultured for 48 hours. Theaccumulated amount of L-threonine was analyzed. According to theanalysis results, two types of the pTHR(*)-containing strains producedthreonine at increased levels of 14% and 22%, respectively, whencompared to the parent strain, E. coli KCCM 10236. In particular, thepTHR(*) vector obtained from the latter strain was designated as pTHR(+)vector. After the pTHR(+) vector was inserted into E. coli w3110(ATCC27325), the obtained strain was designated as E. coli w3110/pTHR(+)and then deposited in the Korean Federation of Culture Collection (KFCC)on Apr. 16, 2002 (accession number: KCCM-10371).

In order to determine the resistance of the recombinant strain, E. coliKCCM 10236(pTHR) to L-isoleucine, the productivity of threonine wasexamined in varying concentration of L-isoleucine-containing media. Theresults are shown in Table 5.

TABLE 3 Solid medium for threonine production Composition ConcentrationGlucose 2% Magnesium sulfate 2 mM Calcium chloride 0.1 mM Disodiumhydrogenphosphate 6 g/L Sodium chloride 0.5 g/L Ammonium chloride 1 g/LPotassium dihydrogenphosphate 3 g/L L-methionine 0.1 g/L L-isoleucine0.1 g/L Yeast extract 10 g/L Agar 18 g/L pH 7.2

TABLE 4 Liquid medium for threonine production Composition ConcentrationGlucose 50 g/L Yeast extract 2 g/L Ammonium sulfate 17 g/L Magnesiumsulfate 1 g/L Sodium chloride 1 g/L Ferrous sulfate 0.01 g/L Manganesesulfate 0.01 g/L L-methionine 0.7 g/L Trace element 1 ml/L L-isoleucine50 mg/L or 200 mg/L Calcium carbonate 30 g/L Potassiumdihydrogenphosphate 0.3 g/L Potassium phsphate, dibasic 0.6 g/L

TABLE 5 Effect of concentration of L-isoleucine on productivity ofL-threonine in E. coli KCCM 10236 containing pTHR(+) amount ofConcentration of Productivity of L-threonine L-isoleucine (g/l)L-threonine (g/l) (%) 0 20.2 40.4 0.05 18.2 36.4 0.10 17.2 34.4 0.2017.5 35.0 0.40 16.5 33.0

As shown in Tables 2 and 5, in the case of the E. coli KCCM10236/pTHR(+), reduction rate of the productivity of L-threonine dependon increase of the concentration of L-isoleucine was considerablydecreased, when compared to the parent strain, E. coli KCCM 10236.Therefore, It can be seen that the pTHR(+) vector contains the mutatedthr operon for L-threonine biosynthesis that is not repressible byL-isoleucine.

Example 5 Identification of Mutation Site

In order to identify mutation site in the thr operon as obtained inExample 4, the nucleotide sequence of the thr operon within the pTHR(+)vector was analyzed. For this, PCR was carried out using 0.1 mg ofpTHR(+) as a template, 2 mM of thr-F (SEQ ID NO: 3) as a primer, and 1μl of BigDye™ Terminator Cycle Sequencing v2.0 Ready Reaction (PEBiosystems). PCR conditions were as follows: denaturation at 95° C. for30 seconds, annealing at 56° C. for 30 seconds, and amplification at 72°C. for 30 seconds. After 30 cycles, the PCR reaction was terminated at4° C. The amplified nucleotide sequence was determined using ABI PRISM3100 Genetic Analyzer (Applied Biosystems). According to the analysisresult, it was found that a 39 bp nucleotide sequence of −56 to −18 thatmaps before the structural thr operon was deleted. The nucleotidesequence of the obtained thr operon is as set forth in SEQ ID NO:2.

The productivity of threonine in the obtained thr operon wasinvestigated. For this, three types of strains, E. coli KCCM 10236, E.coli KCCM 10236/pTHR(+), and E. coli KCCM 10236/pECCG were cultured inflasks containing media for threonine production. The productivities ofthreonine in the strains were evaluated.

In detail, the three strains were cultured overnight in 32° C. incubatorcontaining the media for threonine production of Table 3. One loopful ofeach of the obtained single colonies was inoculated on 25 ml of themedium of Table 4 and cultured for 48 hours at 32° C. and 250 rpm. Theconcentration of the produced L-threonine was measured with HighPerformance Liquid Chromatography (HPLC). The results are presented inTable 6.

TABLE 6 Comparison of threonine production between a parent strain and aselected recombinant strain in a flask Concentration of isoleucineConcentration of threonine (mg/L) Strain (g/L) 50 E. coli KCCM 1023616.9 (parent strain) 16.2 16.5 E. coli KCCM 16.8 10236/pECCG 16.2 16.5E. coli KCCM 19.8 10236/pTHR(+) 20.2 20.5 200 E. coli KCCM 10236 8.76(parent strain) 8.39 9.48 E. coli KCCM 8.15 10236/pECCG 9.06 9.24 E.coli KCCM 16.1 10236/pTHR(+) 16.3 16.6

As shown in Table 6, the productivity of threonine in the recombinantstrain, E. coli KCCM 10236/pTHR(+) was enhanced by 22%, when compared tothe parent strain, E. coli KCCM 10236. In the presence of isoleucine athigh concentration (200 mg/l), the productivity of threonine in theparent strain, E. coli KCCM 10236 was reduced by 46%. On the other hand,the productivity of threonine in the E. coli KCCM 10236/pTHR(+) wasreduced only by 18.9%. Therefore, it can be seen that the strainscontaining the thr operon of the present invention have an increasedresistance to isoleucine repression.

INDUSTRIAL APPLICABILITY

As apparent from the above description, the present invention provides anucleotide sequence of the thr operon of E. coli with a deletion of allor part of a 39 bp nucleotide fragment of −56 to −18 that is attenuatorof the structural genes of the thr operon. In addition, a recombinantvector containing the nucleotide sequence of the thr operon and atransformed host cell containing the recombinant vector are provided.

Therefore, the transformed host cell of the present invention canproduce L-threonine in large scale even in the presence of isoleucine.

1. A nucleotide sequence of the threonine operon of E. coli with adeletion of 39 bp (−56 to −18) of an attenuator as set forth in SEQ IDNO:2.
 2. A recombinant vector containing the nucleotide sequenceaccording to claim
 1. 3. The recombinant vector according to claim 2,wherein a cloning vector for the recombinant vector is pECCG122 obtainedfrom KFCC
 10696. 4. The recombinant vector according to claim 2, whichis pTKR(+).
 5. A transformed host cell containing the recombinant vectoraccording to claim 2, wherein the host cell is maintained in vitro. 6.The transformed host cell according to claim 5, wherein the host cell isEscherichia sp. or Corynebacteria sp.
 7. The transformed host cellaccording to claim 5, wherein the host cell is E.coli KCCM
 10236. 8. Amethod for producing L-threonine comprising: culturing the transformedhost cell according to claim 5; and recovering L-threonine from theculture.